The present invention is generally in the field of biopolymers, and in particular describes a class of polythioesters which can be produced by bacterial fermentation.
Polymers are the most abundant molecules in living matter. There are seven general classes of biopolymers are distinguished: polynucleotides, polyamides, polysaccharides, polyisoprenes, lignin, polyphosphate and polyhydroxyalkanoates, PHA (Mxc3xcller and Seebach, 1993) (Table 1). Poly(3-hydroxybutyrate), PHB, belongs to the latter class as a wide spread bacterial storage compound and was already observed in 1926 as hydrophobic inclusions in the cytoplasm of Bacillus megaterium (Lemoigne, 1926). Today many genera of bacteria are known to accumulate PHAs as energy and carbon source mostly under restricted growth conditions, e.g. oxygen- or nitrogen-limitation (Anderson and Dawes, 1990; Steinbxc3xcchel, 1991).
Polyhydroxyalkanoates (PHAs) are polymers with repeating hydroxy acid monomeric units. PHAs have been reviewed in several publications, including Byrom, xe2x80x9cMiscellaneous Biomaterials,xe2x80x9d in Biomaterials (D. Byrom, ed.) pp. 333-59 (MacMillan Publishers, London 1991); Hocking and Marchessault, xe2x80x9cBiopolyestersxe2x80x9d in Chemistry and Technology of Biodegradable Polymers (G. J. L. Griffin, ed.) pp. 48-96 (Chapman and Hall, London 1994); Mxc3xcller and Seebach, Angew. Chem. Int. Ed. Engl., 32:477-502 (1993); Steinbxc3xcchel, xe2x80x9cPolyhydroxyalkanoic Acids,xe2x80x9d in Biomaterials (D. Byrom, ed.) pp. 123-213 (MacMillan Publishers, London 1991); and Williams and Peoples, CHEMTECH, 26:38-44 (1996).
A wide range of bacteria are known to accumulate polyhydroxyalkanoates (PHA) as intracellular storage compounds. Due to the properties of these polymers as biodegradable thermoplastics, and elastomers they have attracted much interest and are considered for various technical applications in industry, medicine, agriculture and other areas (Anderson, A. J. and Dawes, E. A., Microbiol. Rev. 54, 450-472 (1990); Steinbxc3xcchel, 1991).
Several types of polyhydroxyalkanoates are formed in nature by various organisms in response to environmental stress. These PHAs can be broadly divided into three groups according to the length of their pendant groups and their respective biosynthetic pathways. Relatively short pendant groups include the C3-5 hydroxy acids, whereas relatively long pendant groups include C6-14 hydroxy acids.
There are three major types of naturally occurring PHAs. The first type includes only relatively short hydroxy acid monomeric units. The second type include both relatively short and relatively long hydroxy acid monomeric units. The third type includes only relatively long hydroxy acid monomeric units. Those with short pendant groups, such as polyhydroxybutyrate (PHB), a homopolymer of R-3-hydroxybutyric acid (R-3HB) units, are highly crystalline thermoplastic materials (Lemoigne and Roukhelman, Annales des fermentations, 5:527-36 (1925)). PHAs containing the short R-3HB units randomly polymerized with much longer pendant group hydroxy acid units were first reported in the early seventies (Wallen and Rohwedder, Environ. Sci. Technol., 8:576-79 (1974)). A number of microorganisms which specifically produce copolymers of R-3HB with these longer pendant group hydroxy acid units are also known and belong to this second group (Steinbxc3xcchel and Wiese, Appl. Microbiol Biotechnol., 37:691-97 (1992)). In the early 1980""s, a research group in The Netherlands identified the third group of PHAs, which contains predominantly longer pendant group hydroxy acids (De Smet, et al., J. Bacteriol., 154:870-78 (1983)).
PHAs may constitute up to 90% of the dry cell weight of bacteria, and are found as discrete granules inside the bacterial cells. These PHA granules accumulate in response to nutrient limitation and serve as carbon and energy reserve materials. Distinct pathways are used by microorganisms to produce each group of these polymers. One of these pathways leading to the short pendant group polyhydroxyalkanoates (SPGPHAs) involves three enzymes: thiolase, reductase, and PHB synthase (sometimes called polymerase). Using this pathway, the homopolymer PHB is synthesized by condensation of two molecules of acetyl-Coenzyme A to give acetoacetyl-Coenzyme A, followed by reduction of this intermediate to R-3-hydroxybutyryl-Coenzyme A, and subsequent polymerization. The last enzyme in this pathway, the synthase, has a substrate specificity that can accommodate C3-5 monomeric units, including R-4-hydroxy acid and R-5-hydroxy acid units. This biosynthetic pathway is found, for example, in the bacteria Zoogloea ramigera and Alcaligenes eutrophus. 
The biosynthetic pathway which is used to make the third group of PHAs, long pendant group polyhydroxyalkanoates (LPGPHAs), is still partly unknown. However, it is currently thought that the monomeric hydroxyacyl units leading to the LPGPHAs are derived by the xcex1-oxidation of fatty acids and the fatty acid pathway. The R-3-hydroxyacyl-Coenzyme substrates resulting from these routes are then polymerized by PHA synthases (sometimes called polymerases) that have substrate specificities favoring the larger monomeric units in the C6-14 range. LPGPHAs are produced, for example, by Pseudomonads.
The second group of PHAs containing both short R-3HB units and longer pendant group monomers are believed to utilize both the pathways to provide the hydroxy acid monomers. The latter are then polymerized by PHA synthases able to accept these units.
Roughly 100 different types of PHAs have been produced by fermentation methods so far (Steinbxc3xcchel and Valentin, FEMS Microbiol., Lett., 128:219-28 (1995)). A number of these PHAs contain functionalized pendant groups such as esters, double bonds, alkoxy, aromatic, halogens, and hydroxy groups. Transgenic systems for producing PHAs in both microorganism and plants, as well as enzymatic methods for PHA synthesis, are reviewed by Williams and Peoples, CHEMTECH, 26:38-44 (1996).
Two PHAs belonging to the first group, polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV), have been extensively studied. PHBV is a copolymer of R-3HB units with 5-24% R-3-hydroxyvaleric acid (R-3HV), and is known commercially as Biopol(trademark) (supplied by ICI/Zeneca). These polymers are natural thermoplastics which can be processed using conventional polymer technology and which have industrially useful properties, such as biodegradability in soil and marine environments and good barrier properties. They are characterized by melting points which range from 130 to 180xc2x0 C., and extensions-to-break of 8 to 42% (see Zeneca Promotional Literature, Billingham, UK 1993).
So far, more than 130 different hydroxyalkanoic acids have been described as constituents of PHAs, comprising 3-, 4-, 5-, and 6-hydroxyalkanoic acids of various chain length. The pendant alkyl side chain can in addition contain various constituents (for review see Steinbxc3xcchel and Valentin, 1995). Whereas the large variety of PHA constituents refers almost exclusively to the modified side chains in the xcex2-position of the hydroxyalkanoic acids, PHAs with modified backbones are rare. Examples are 2-methyl-3-hydroxybutyric acid and 3-hydroxypivalic acid, which have been identified as PHA constituents resulting in polymer chains with one or two methyl groups, respectively, in the backbone (Satho et al., (1992) Wat. Sci. Technol. 26, 933-942; Fxc3xcchtenbusch et al., (1998) FEMS Microbiol. Lett. 159, 85-92).
Bacteria synthesize PHAs from coenzyme A thioesters of the respective hydroxyalkanoic acid and are able to produce a wide range of different PHAs due to the rather unspecific PHA synthases that catalyze the polymerization reaction. In 1974, 3-hydroxyvaleric acid and 3-hydroxyhexanoic acid were identified as additional constituents of these bacterial polyesters (Wallen and Rohwedder, 1974). Only a few polyesters can be obtained from simple and abundantly available carbon sources, e.g. glucose. The large variety of PHAs comprises 3-, 4-, 5-, and 6-hydroxyalkanoic acids of varying chain length, possibly containing additional methyl or other alkyl groups, double bonds, or different substituents at various positions of the hydroxyalkanoic acid and is often based on the feeding of suitable precursor substrates, which exhibit chemical structures related to the PHA constituents (Steinbxc3xcchel and Valentin, 1995).
It is an object of the present invention to provide a class of polyhydroxyalkanoates which include a thioester bond in the polymer backbone or a thioether bond in the polymer side chains.
It is a further object of the present invention to provide a means for producing polyhydroxyalkanoates which include a thioester bond or thioether bond.
Biopolymers including sulfur in the form of a thioester in the polymer backbone or a thioether in the polymer side chains have been developed. These are preferably produced by fermentation of bacteria with appropriate sulfur containing substrates, which are incorporated by a broad spectrum PHA polymerase.
As demonstrated by example 1, a hitherto unknown copolymer that contains sulfur in the backbone linking 3-mercaptopropionic acid and 3-hydroxybutyric acid by thioester linkages was synthesized by R. eutropha. Besides proteins and some complex polysaccharides, this is the first demonstration of the biosynthesis of a polymer containing sulfur. The copolymer contributed up to 19% of the cellular dry weight and consisted of up to 43 mol% of 3-mercaptopropionic acid.
As demonstrated by example 2, a hitherto unknown copolymer that contains sulfur in the backbone linking 3-hydroxybutyrate and 3-mercaptobutyrate by thioester linkages was synthesized by the polyhydroxyalkanoate- (PHA) accumulating bacterium R. eutropha, when 3-mercaptobutyric acid was fed as carbon source in addition to gluconate. The chemical structure of this polymer was confirmed by gas chromatography/mass spectrometry, infrared spectroscopy, 1H- and 13C-nuclear magnetic resonance spectroscopy, and elemental sulfur analysis.
As demonstrated by example 3, in the presence of PTO in the medium, R. eutropha PHB-4 pBBR1 : phaC 1 synthesized a hitherto unknown polyester including exclusively 3-hydroxypropylthiobutyrate, 3-hydroxypropylthiohexanoate, and 3-hydroxypropylthiooctanoate as polymer constituents, poly(3HPTB-co-3HPTHx-co-3HPTO). Larger amounts of poly(3HPTB-co-3HPTHx-co-3HPTO) can also be produced via biological engineering.
The sulfur-containing PHAs allow various applications and uses in industry. Representative embodiments of the applications of the sulfur-containing PHAs include their uses in the packaging industry, medicine, pharmacy, agriculture or food industry, as active agents or as coatings, packaging, or carriers.
In one preferred embodiment, the sulfur-containing PHAs can be used as an anti-bacterial agent, an anti-viral agent, or an anti-fungal agent. In one most preferred embodiment, the sulfur-containing PHAs can be used as anti-bacterial coatings.
In another preferred embodiment, the sulfur-containing PHAs are used as electrolytes.